Circadian clocks have been described in organisms ranging in complexity from unicells to mammals and function to control daily rhythms in cellular activities and behavior. The significance of a detailed understanding of the clock can be appreciated by its ubiquity and its established involvement in human physiology including endocrine function, sleep/wake cycles, psychiatric illness, as well as drug tolerances and effectiveness. Additionally, cell division in many human tissues is clock-regulated, providing the basis for promising new approaches to cancer chemotherapy. Our long term goals are to understand the molecular and biochemical basis for circadian rhythmicity. The clock in all organisms is assembled within the cell and clock components are evolutionarily conserved; thus, simple eukaryotes provide appropriate experimental systems to investigate clock mechanisms and to efficiently achieve these goals. An important aspect of circadian rhythmicity is clock control of gene expression. However, little is known about how this regulation takes place or of the components that signal time information in the cell. To answer these questions, we are focusing our studies on the biochemical function and regulation of clock-controlled genes in the model system Neurospora crassa. In Specific Aim 1 we will use biochemical techniques to isolate the trans-acting factor(s) that bind to a positive cis-acting clock element in the ccg-2 gene which is both necessary and sufficient for rhythmicity. The gene(s) encoding the factor(s) will be cloned and analyzed with respect to their role in circadian output pathways. In Specific Aim 2 we will carry out a genetic mutant selection for novel genes involved in the regulation of circadian output based on differential expression of the clock-controlled genes in a wild type versus a clock-null strain. Fusion of the promoter of two clock-controlled genes, one positively and one negatively regulated by a pathway involving FRQ, to the selectable marker mtr will permit the isolation of mutants that result in improper expression of the chimera. In Specific Aim 3 we will use a brute force screen for novel clock output signaling mutants, as well as for mutants in environmental input pathways to the clock and the clock itself. The loci identified in the mutant selections and screens will be assayed for their role in circadian rhythmicity, cloned, and important genes will be used to initiate a search for mammalian orthologs as described in Specific Aim 4. Together, these experiments will permit a more detailed understanding of how the cell is organized as a function of time.